Bridge-helix cap: target and method for inhibition of bacterial rna polymerase

ABSTRACT

It has been discovered that the Sal target represents a new and promising target for antibacterial drug discovery. The Sal target is distinct from the rifamycin target and from the CBR703 target. This indicates that antibacterial compounds that function through the Sal target should exhibit no, or minimal, cross-resistance with rifamycins and CBR703. This further implies that it should be possible to co-administer antibacterial compounds that function through the Sal target together with a rifamycin, together with CBR703, or together with both a rifamycin and CBR703, in order to achieve additive or synergistic antibacterial effects and in order to suppress or eliminate the emergence of resistance.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application is a divisional of U.S. application Ser. No.14/006,035, which is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2012/029679, filed Mar. 19, 2012, which claims thebenefit of U.S. Provisional Application Ser. No. 61/454,323, filed Mar.18, 2011, now expired. The entire content of the applications referencedabove are hereby incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R01-GM41376 andR01-A172766 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, is named 83503WO1.txt and is29,392 bytes in size.

BACKGROUND

Bacterial infections remain among the most common and deadly causes ofhuman disease. Multi-drug-resistant bacteria now cause infections thatpose a grave and growing threat to public health. It has been shown thatbacterial pathogens can acquire resistance to first-line and evensecond-line antibiotics. New approaches to drug development for treatingbacterial infections are necessary, e.g., to combat the ever-increasingnumber of antibiotic-resistant pathogens.

SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

We disclose here that the antibiotic salinamide A (Sal) inhibitsbacterial RNA polymerase (RNAP) in vivo, that Sal kills bacteria byinhibiting RNAP, and how Sal inhibits RNAP. To determine whether Salinhibits RNAP in vivo, effects of Sal on RNAP activity in vivo wereassayed. Upon addition of Sal to bacteria, RNAP-dependent RNA synthesisdecreased immediately, indicating that Sal inhibits RNAP in vivo. Todetermine whether Sal kills bacteria by inhibiting RNAP, spontaneousSal-resistant mutants of were isolated, and genes for RNAP subunits inSal-resistant mutants were amplified and sequenced. One hundred percent(39 of 39) of analyzed Sal-resistant mutants contained mutations ingenes encoding RNAP subunits, indicating that Sal kills bacteria byinhibiting RNAP. Substitutions conferring Sal-resistance were obtainedat positions 690, 697, 738, 748, 758, 763, 779, 780, 782, and 783 ofEscherichia coli RNAP subunit and positions 569, 675, and 677 ofEscherichia coli RNAP β-subunit. Additional Sal-resistant mutants wereisolated following mutagenesis of genes encoding RNAP subunits. In theseadditional Sal-resistant mutants, substitutions conferringSal-resistance were identified at positions 758, 780, and 782 ofEscherichia coli RNAP (3′ subunit and positions 561, 665, and 680 ofEscherichia coli RNAP β subunit.

When mapped onto the structure of RNAP, the sites of substitutionsconferring Sal-resistance formed a compact cluster. We designate thesites of substitutions conferring Sal-resistance as the “Sal target.”

Determination of a crystal structure of Escherichia coli RNAP in complexwith Sal showed that the Sal target is the binding site on RNAP for Sal,and that sites of substitutions conferring Sal-resistance correspond toRNAP residues of RNAP that contact, or are close to, Sal.

The Sal target does not overlap the targets of currently usedantibiotics. Accordingly, Sal does not exhibit cross-resistance withcurrently used antibiotics, and coadministration of Sal with a currentlyused antibiotic results in an extremely low spontaneous resistancefrequency. The Sal target represents an attractive new target forantibacterial drug discovery.

A subset of residues of the Sal target have no overlap with anypreviously described target of any previously described RNAP inhibitor:i.e., residues 690, 697, 758, and 763 of Escherichia coli RNAP β′subunit and residues 561, 569, 665, 675, 677, and 680 of Escherichiacoli RNAP β subunit. In the structure of RNAP, these residues arelocated in proximity to the N-terminus of the RNAP-active-center bridgehelix, in and near RNAP-active-center sub-regions termed the “β′ Floop,” the “β D2 loop,” and the “β link region” (FIGS. 4A-C). Wedesignate these residues as the “bridge-helix cap target.”

In RNAP from Escherichia coli, the bridge-helix cap target comprisesresidues 690, 697, 758, and 763 of Escherichia coli RNAP β′ subunit andresidues 561, 569, 665, 675, 677, and 680 of Escherichia coli RNAP βsubunit. In RNAP from other bacterial species, the bridge-helix captarget comprises the residues corresponding to, and alignable with,residues 690, 697, 758, and 763 of Escherichia coli RNAP β′ subunit andresidues 561, 569, 665, 675, 677, and 680 of Escherichia coli RNAP βsubunit (FIGS. 4A-C). For example, in RNAP from Bacillus subtilis, thebridge-helix cap target comprises the residues corresponding to, andalignable with, residues 709, 716, 762, and 767 of Bacillus subtilisRNAP β′ subunit and residues 517, 525, 623, 633, 635, and 638 ofBacillus subtilis RNAP β subunit (FIGS. 4A-C).

Accordingly, certain embodiments of the present invention provide

(i) a target for binding of a molecule to an RNA polymerase from abacterial species, comprising at least one residue corresponding to, andalignable with, residues 561, 569, 665, 675, 677, and 680 of the βsubunit and residues 690, 697, 758, and 763 of the β′ subunit of RNApolymerase from Escherichia coli (“bridge-helix cap target”);

(ii) a target for inhibition by a molecule of an RNA polymerase from abacterial species, comprising at least one residue corresponding to, andalignable with, residues 561, 569, 665, 675, 677, and 680 of the βsubunit and residues 690, 697, 758, and 763 of the β′ subunit of RNApolymerase from Escherichia coli (“bridge-helix cap target”);

(iii) a method to identify a molecule that binds to the target of i andii comprising identification of a molecule that (a) binds to an RNApolymerase from a bacterial species, but (b) does not bind, or bindsless well, to a derivative of an RNA polymerase from a bacterial speciesthat has at least one amino acid substitution, deletion, or insertion,in the set of residues taught in i and ii;

(iv) a method to identify a molecule that inhibits an RNA polymerasefrom a bacterial species through the target of i and ii, comprisingidentification of a molecule that (a) inhibits an RNA polymerase from abacterial species, but (b) does not inhibit, or inhibits less well, aderivative of an RNA polymerase from a bacterial species that has atleast one amino acid substitution, deletion, or insertion, in the set ofresidues taught in i and ii;

(v) a method to identify a molecule that inhibits growth of a bacteriumthrough the target of i and ii, comprising identification of a moleculethat (a) inhibits growth of a bacterium, but (b) does not inhibit, orinhibits less well, a derivative of said bacterium that contains aderivative of an RNA polymerase that has at least one amino acidsubstitution, deletion, or insertion, in the set of residues taught in iand ii; and

(vi) a method to identify a molecule that binds to an RNA polymerasefrom a bacterial species through the target of i and ii, comprising (a)preparation of a first molecule that binds to the target of i and ii andthat contains a detectable group, and (b) identification of a secondmolecule that competes with said first molecule for binding to an RNApolymerase from a bacterial species.

(vii) a method to treat a bacterial infection in a subject in needthereof, comprising administering to the subject a first compoundselected as being an inhibitor of growth of a bacterium by binding tothe target of i and ii, and a second compound that inhibits growth of abacterium by binding to a site other than the target of i and ii.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of Sal.

FIGS. 2A-B. Measurement of effects of Sal on nucleic acid synthesis invivo. (FIG. 2A) Experimental approach. (FIG. 2B) Effects of Sal on RNA(left) and DNA (right) synthesis in vivo.

FIGS. 3A-C. Isolation of spontaneous Sal-resistant mutants, followed byPCR-amplification and sequencing of RNAP-subunit genes. (FIG. 3A)Experimental approach. (FIG. 3B) Representative data, isolation ofSal-resistant mutants. (FIG. 3C) Representative data, PCR-amplificationof rpoC (left) and rpoB (right) genes.

FIGS. 4A-C. Sal target: location in the amino acid sequence of RNAP β′and ρ subunits. (FIG. 4A, FIG. 4B and FIG. 4C) Amino acid sequencealignments for regions of E. coli RNAP β′ subunit and β subunitcontaining single-residue substitutions that confer Sal-resistance (seeTables 2-3). Sequences for bacterial RNAP are in the top twenty rows ofFIG. 4A, FIG. 4B and FIG. 4C (Sequences for bacterial RNAP are disclosedas SEQ ID NOS 5-64, respectively, in order of appearance. Sequences forbacterial RNAP are disclosed as SEQ ID NOS 74-113, respectively, inorder of appearance.); sequences for human RNAP I, RNAP II, and RNAP IIIare in the bottom three rows of FIG. 4A, FIG. 4B and FIG. 4C (Sequencesfor human RNAP I, RNAP II, and RNAP III are disclosed as SEQ ID NOS65-73, respectively, in order of appearance. Sequences for human RNAP I,RNAP II, and RNAP III are disclosed as SEQ ID NOS 114-119, respectively,in order of appearance.); sites of single-residue substitutions thatconfer Sal-resistance are boxed (with E. coli residue numbers); RNAPactive-center sub-regions are indicated with black bars above thesequences. Species are as follows: E. coli (ECOLI), Haemophilusinfluenzae (HAEIN), Vibrio cholerae (VIBCH), Pseudomonas aeruginosa(PSEAE), Treponema pallidum (TREPA), Bordetella pertussis (BORPE),Xylella fastidiosa (XYLFA), Campylobacter jejuni (CAMJE), Neisseriameningitidis (NEIME), Rickettsia prowazekii (RICPR), Chlamydiatrachomatis (CHLTR), Mycoplasma pneumoniae (MYCPN), Bacillus subtilis(BACSU), Staphylococcus aureus (STAAU), Mycobacterium tuberculosis(MYCTU), Synechocystis sp. PCC 6803 (SYNY3), Aquifex aeolicus (AQUAE),Deinococcus radiodurans (DEIRA), Thermus aquaticus (THEAQ), Thermusthermophilus (THETH), and Homo sapiens (HUMAN).

FIG. 5. Sal target: location in the three-dimensional structure of RNAP.Three-dimensional structure of RNAP showing the Sal target. RNAP isillustrated in a ribbon representation in gray. Sites of substitutionsthat confer Sal-resistance (see Tables 2-3) are illustrated in aspace-filling representation. (*) The RNAP active-center Mg²⁺ ion isillustrated as a sphere (**). The structure shown is a crystal structureof T. thermophilus RNAP holoenzyme (8; (3′-subunit non-conserved regionand a subunit omitted for clarity); correspondences between residues ofE. coli RNAP and T. thermophilus RNAP are based on amino acid sequencealignments (see FIGS. 4A-C).

FIG. 6. Sal target: relationship between the Sal target, the rifamycintarget, and the CBR703 target. Three-dimensional structure of RNAPshowing the Sal target, the rifamycin target, and the CBR703 target.Sites of substitutions that confer Sal-resistance (*) (see Tables 2-3),rifamycin-resistance (***) (10-15), and CBR703-resistance (****) (16,17)are illustrated in space-filling representations. The RNAP active-centerMg²⁺ ion is illustrated as a sphere (**).

FIGS. 7A-B. Crystal structure of E. coli RNAP σ⁷⁰ holoenzyme in complexwith Sal. FIG. 7A shows electron density for Sal (#; 2σ F_(o)-F_(c)difference electron density for E. coli RNAP σ⁷⁰ holoenzyme with Sal vs.E. coli RNAP holoenzyme without Sal). FIG. 7B shows, for comparison,sites of substitutions that confer Sal-resistance (*; see Tables 2-3).In each panel, the RNAP active-center Mg²⁺ ion is illustrated as asphere (**).

DETAILED DESCRIPTION

It has been discovered that the bridge-helix cap target represents a newand promising target for antibacterial drug discovery. The bridge-helixcap target is distinct from the rifamycin target and from the CBR703target. This implies that antibacterial compounds that function throughthe bridge-helix cap target should exhibit no, or minimal,cross-resistance with rifamycins and CBR703. This further implies thatit should be possible to co-administer antibacterial compounds thatfunction through the bridge-helix cap target together with a rifamycin,together with CBR703, or together with both a rifamycin and CBR703, inorder to achieve additive or super-additive/synergistic antibacterialeffects and in order to suppress or eliminate the emergence ofresistance.

Accordingly, certain embodiments of the present invention provide amethod to identify a molecule that binds to the bridge-helix cap targetof an RNA polymerase from a bacterial species, comprising identifying amolecule that binds to an RNA polymerase from a bacterial species butbinds substantially less to a derivative of an RNA polymerase from abacterial species that has at least one amino acid substitution,deletion, or insertion of at least one residue corresponding to, oralignable with, residues 561, 569, 665, 675, 677, or 680 of the βsubunit or residues 690, 697, 758, or 763 of the β′ subunit of RNApolymerase from Escherichia coli.

Certain embodiments of the present invention provide a method toidentify a molecule that inhibits an RNA polymerase from a bacterialspecies by binding to the bridge-helix cap target of the RNA polymerase,comprising identifying a molecule that inhibits an RNA polymerase from abacterial species but inhibits substantially less a derivative of an RNApolymerase from a bacterial species that has at least one amino acidsubstitution, deletion, or insertion of at least one residuecorresponding to, or alignable with, residues 561, 569, 665, 675, 677,or 680 of the β subunit or residues 690, 697, 758, or 763 of the β′subunit of RNA polymerase from Escherichia coli.

Certain embodiments of the present invention provide a method toidentify a molecule that inhibits growth of a bacterium, comprisingidentifying a molecule that inhibits growth of a bacterium but inhibitsgrowth substantially less of a derivative of said bacterium that has atleast one amino acid substitution, deletion, or insertion of at leastone residue corresponding to, or alignable with, residues 561, 569, 665,675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 ofthe β subunit of RNA polymerase from Escherichia coli.

Certain embodiments of the present invention provide a method toidentify a molecule that binds to an RNA polymerase from a bacterialspecies through the bridge-helix cap target of the RNA polymerase,comprising identifying a target molecule that competes with a firstmolecule for binding to an RNA polymerase from a bacterial species,wherein the first molecule preferentially binds to the bridge-helix captarget of the RNA polymerase.

In certain embodiments, the first molecule comprises a detectable group.

Certain embodiments of the present invention provide a method to treat abacterial infection in a subject in need thereof, comprisingadministering to the subject a first compound selected as being aninhibitor of growth of a bacterium by binding to the bridge-helix captarget of an RNA polymerase, and a second compound that inhibits growthof a bacterium by binding to a site other than the bridge-helix captarget of an RNA polymerase.

In certain embodiments, the first compound is salinamide A.

In certain embodiments, the second compound is a rifamycin or CBR703.

In certain embodiments, the method further comprises administering athird compound that inhibits growth of a bacterium by binding to a siteother than the bridge-helix cap target of an RNA polymerase.

In certain embodiments, the first and second compounds are administeredconcurrently.

In certain embodiments, the first and second compounds are administeredsequentially.

In certain embodiments, the third compound is administered concurrently.

In certain embodiments, the third compound is administered sequentially.Certain embodiments of the present invention provide a composition(e.g., a pharmaceutical composition), or a kit, that comprisessalinamide A and a rifamycin and/or CBR703.

In certain embodiments, the composition or kit comprises salinamide Aand a rifamycin.

In certain embodiments, the composition or kit comprises salinamide Aand CBR703.

In certain embodiments, the composition or kit comprises salinamide A, arifamycin and CBR703.

Certain embodiments of the present invention provide a method to treat abacterial infection in a subject in need thereof, comprisingadministering to the subject a composition described herein.

Certain embodiments of the present invention provide the use of acomposition described herein to treat a bacterial infection.

Certain embodiments of the present invention provide an RNA polymerase,e.g., an isolated RNA polymerase, that has at least one amino acidsubstitution, deletion, or insertion of at least one residuecorresponding to, or alignable with, residues 561, 569, 665, 675, 677,or 680 of the β subunit or residues 690, 697, 758, or 763 of the β′subunit of RNA polymerase from Escherichia coli.

Certain embodiments of the present invention provide a bacterium, e.g.,an isolated bacterium, that comprises an RNA polymerase that has atleast one amino acid substitution, deletion, or insertion of at leastone residue corresponding to, or alignable with, residues 561, 569, 665,675, 677, or 680 of the β subunit or residues 690, 697, 758, or 763 ofthe β′ subunit of RNA polymerase from Escherichia coli.

Provided herein are targets and methods for specific binding andinhibition of RNA polymerase from bacterial species. Embodiments of theinvention have applications in control of bacterial gene expression,control of bacterial growth, antibacterial chemistry, and antibacterialtherapy.

Certain embodiments of the invention include: a new target (thebridge-helix cap target) and associated assay methods for inhibition ofbacterial RNA polymerase, inhibition of bacterial RNA synthesis andinhibition of bacterial growth. Antibacterial compounds that functionthrough the new target should exhibit minimal or no cross-resistancewith antibacterial compounds that function through previously disclosedtargets. It should be possible to co-administer antibacterial compoundsthat function through the new target together with antibacterialcompounds that function through previously disclosed targets, to achieveadditive antibacterial effects and to suppress the emergence ofresistance.

Certain embodiments of the invention will now be illustrated by thefollowing non-limiting Example.

Example 1 Identification of the Target of the Antibiotic Salinamide A

Salinamide A (Sal) is a bicyclic depsipeptide antibiotic, comprisingseven amino acids and two non-amino-acid residues (1,2; FIG. 1). Sal isproduced by Streptomyces sp. CNB-091, a marine bacterium isolated fromthe surface of the jellyfish Cassiopeia xamachana (1-3), and also byStreptomyces sp. NRRL 21611, a soil bacterium (4). Sal exhibitsantibacterial activity against bacterial pathogens includingStreptococcus pneumoniae and S. pyogenes (1,2).

Sal has been reported to inhibit bacterial RNA polymerase (RNAP), theenzyme responsible for bacterial RNA synthesis, in vitro (IC₅₀=0.5 μM;4). However, it has not previously been determined whether RNAP is thefunctional cellular target of Sal.

The hypothesis of this research was that RNAP is the functional cellulartarget of Sal. Corollaries to this hypothesis were that Sal shouldinhibit RNAP in vivo, that mutations conferring resistance to Sal shouldoccur in RNAP-subunit genes, and that mapping of sites of substitutionsconferring resistance to Sal onto the three-dimensional structure ofRNAP should define residues of RNAP important for function of Sal. Theobjectives of this research were to determine whether Sal inhibits RNAPin vivo, to determine whether Sal kills bacteria by inhibiting RNAP, andto determine how Sal inhibits RNAP.

The results of this research show that Sal inhibits RNA synthesis invivo (FIGS. 2A-B) and show that mutations in RNAP-subunit genes resultin Sal-resistance (FIGS. 3A-C; Tables 1-3), thereby demonstrating thatRNAP is the functional cellular target of Sal. In addition, the resultsof this research show that transcription inhibition by Sal requires acompact determinant—the “Sal target”—adjacent to and overlapping theRNAP active center and adjacent to but not overlapping the targets ofthe RNAP inhibitors rifamyins and CBR703 (FIG. 5; Tables 2-5). The Saltarget comprises RNAP β′-subunit residues 690, 697, 738, 748, 758, 763,775, 779, 780, 782, and 783 and RNAP β-subunit residues 561, 569, 665,675, 677, and 680. Finally, determination of a crystal structure of E.coli RNAP in complex with Sal shows that the Sal target is the bindingsite on RNAP for Sal, and that sites of substitutions conferringSal-resistance correspond to RNAP residues of RNAP that contact or areclose to Sal (FIGS. 7A-B).

The Sal target is located adjacent to, and partly overlaps, the RNAPactive center (FIGS. 4A-C, 5, 6 and 7A-B). It is inferred that Sal mostlikely inhibits RNAP by inhibiting RNAP active-center function.

Most sites of substitutions conferring Sal-resistance involve aminoacids that are conserved in RNAP from a broad range of bacterial species(FIGS. 4A-C, upper rows in each panel); this is consistent with, andaccounts for, the observation that Sal inhibits RNAP from a broad rangeof bacterial species (4). Nine of the sites of substitutions conferringSal-resistance—β′ residues 690, 697, 738, 775 and 779, and β residues569, 675, 677, and 680—are not conserved in human RNAP I, RNAP II, andRNAP III (FIGS. 4A-C, bottom three rows in each panel); this isconsistent with, and accounts for, the observation that Sal does notinhibit human RNAPs (4).

Mapping of substitutions conferring Sal-resistance onto thethree-dimensional structure of a transcription elongation complexcomprising RNAP, DNA, RNA, and a nucleoside triphosphate (9) indicatesthat the Sal target does not overlap the RNAP active-center Mg²⁺ ion anddoes not overlap RNAP residues that interact with the DNA template, theRNA product, and the nucleoside triphosphate substrate. It is inferredSal most likely inhibits RNAP active-center function allosterically,through effects on RNAP conformation, and not through directinteractions with RNAP residues that mediate bond formation, productbinding, and substrate binding.

The Sal target overlaps RNAP active-center sub-regions that have beendesignated as the “μ′ bridge helix hinge N” (BH-H_(N)), the “β′ F loop,”the “β link region,” and the “β D2 loop” (FIGS. 4A-C; active-centersub-region nomenclature as in 18,19). Fully 20 of the 24 identifiedsubstitutions conferring Sal-resistance map to these RNAP active-centersub-regions (FIGS. 4A-C; Tables 2-3). The BH-H_(N) may undergoconformational changes coupled to, and essential for, thenucleotide-addition cycle in RNA synthesis, and the F-loop, and possiblyalso the link region and the D-2-loop, may coordinate theseconformational changes (18,19). It is inferred herein that Sal mostlikely inhibits RNAP active-center function by inhibiting BH-H_(N)hinge-opening and/or hinge-closing.

Sal is the first RNAP inhibitor that has been inferred to functionthrough effects on BH-H_(N) conformational cycling. Accordingly, Salwill find use as a research tool for dissection of mechanistic andstructural aspects of BH-H_(N) conformational cycling.

Relationship Between the Sal Target and the Rifamycin Target.

The Sal target is located adjacent to, but does not overlap, the targetof the rifamycin antibacterial agents (e.g., rifampin, rifapentine,rifabutin, and rifalazil), which are RNAP inhibitors in current clinicaluse in antibacterial therapy (FIG. 6). Consistent with the lack ofoverlap between the Sal target and the rifamycin target, Sal-resistantmutants are not cross-resistant to the rifamycin rifampin (Table 4), andrifamycin-resistant mutants are not cross-resistant to Sal (Table 5).The absence of overlap and the absence of cross-resistance indicatesthat it should be possible to co-administer Sal and a rifamycin in orderto achieve additive or superadditive antibacterial activities and inorder to suppress the emergence of resistance. Consistent with thisinference, co-administration of Sal and the rifamycin rifampin resultsin an extremely low, effectively negligible, spontaneous resistancefrequency: <2×10⁻¹¹(<1/200 the spontaneous resistance frequency of Salalone; <1/500 the spontaneous resistance frequency of rifampin alone;Table 6). This is important in view of the fact that susceptibility tospontaneous resistance is the main limiting factor in the clinical useof rifamycins (15,21,22).

Relationship Between the Sal Target and the CBR703 Target.

The Sal target also is located adjacent to, but does not overlap, thetarget of CBR703, an RNAP inhibitor under investigation for clinical usein antibacterial therapy (FIG. 6). Consistent with the lack of overlapbetween the Sal target and the CBR703 target, Sal-resistant mutantsexhibit no cross-resistance to CBR703 (Table 4). In fact, the majorityof Sal-resistant mutants exhibit hypersensitivity to CBR703, and halfexhibit high-level hypersensitivity to CBR703 (i.e., are at least fourtimes more sensitive to CBR703 than the wild-type parent strain; Table4). The absence of overlap, the absence of cross-resistance, and thepresence of hypersensitivity, indicate that it should be possible toco-administer Sal and CBR703 in order to achieve additive orsuperadditive antibacterial activities and in order to suppress theemergence of resistance. Consistent with this inference,co-administration of Sal and CBR703 results in an extremely low,effectively negligible, spontaneous resistance frequency: <2×10⁻¹¹(<1/200 the spontaneous resistance frequency of Sal alone; <1/10 thespontaneous resistance frequency of CBR703 alone; Table 7).

Results

Sal Inhibits RNA Synthesis In Vivo.

To determine whether Sal inhibits RNAP in vivo, effects of Sal on RNAsynthesis in vivo were assayed, and, as a control, effects of Sal on DNAsynthesis in vivo were assayed. Sal was added to cultures of E. coliD21f2tolC growing in media containing either a radioactively labeledRNA-synthesis precursor ([¹⁴C]-uracil) or a radioactively labeled DNAsynthesis precursor ([¹⁴C]-thymidine); aliquots were removed and mixedwith trichloroacetic acid (TCA) 0, 5, 10, and 15 min thereafter to lysecells and precipitate nucleic acids; TCA-precipitated nucleic acids werecollected by vacuum filtration; and radioactivity in TCA-precipitatednucleic acids was quantified (FIG. 2A). It was observed that, uponaddition of Sal to bacterial cultures, RNA synthesis essentially stopped(FIG. 2B, left panel). The effect was rapid; it was observed at theearliest time point tested (5 min; FIG. 2B, left panel). The effect wasspecific; addition of Sal to bacterial cultures had no effect on DNAsynthesis (FIG. 2B, right panel). It is concluded that Sal inhibits RNAsynthesis in vivo, and it is inferred that Sal inhibits RNAP in vivo.

Sal-Resistant Mutants Map to RNAP-Subunit Genes.

To determine whether Sal kills bacteria by inhibiting RNAP, spontaneousSal-resistant mutants were isolated, and genes for RNAP subunits inSal-resistant mutants were PCR-amplified and sequenced (FIG. 3A).Spontaneous Sal-resistant mutants were isolated by plating high-densitycultures of E. coli D21f2tolC (˜3×10⁹ cells per plate) on agarcontaining Sal and identifying rare resistant colonies (FIG. 3B). Foreach Sal-resistant mutant, genomic DNA was prepared, and the genes forthe largest RNAP subunit and the second-largest RNAP subunit—rpoCencoding RNAP β′ subunit and rpoB encoding RNAP β subunit werePCR-amplified and sequenced (FIG. 3C).

Spontaneous Sal-resistant mutants were isolated with a frequency of˜4×10⁻⁹ (Table 1). A total of 39 Sal-resistant mutants were isolated,PCR-amplified, and sequenced (Table 1). Strikingly, one hundred percent(39 of 39) of sequenced Sal-resistant mutants were found to containmutations in genes for RNAP subunits: 31 were found to contain singlemutations in rpoC, 1 was found to contain a double mutation in rpoC, and7 were found to contain single mutations in rpoB (Table 1). It isconcluded that a single substitution in an

RNAP-subunit gene, either rpoC or rpoB, is sufficient to conferSal-resistance, and it is inferred that RNAP is the functional cellulartarget for Sal.

TABLE 1 Sal-resistant mutants: summary statistics frequency ofspontaneous mutation to Sal-resistance ~4 × 10⁻⁹ number of Sal-resistantisolates 39 number of Sal-resistant isolates containingsingle-substitution 31 mutations in rpoC number of Sal-resistantisolates containing multiple- 1 substitution mutations in rpoC number ofSal-resistant isolates containing single-substitution 7 mutations inrpoB number of Sal-resistant isolates containing multiple- 0substitution mutations in rpoB percentage of Sal-resistant isolatescontaining mutations in 100 RNAP-subunit genes

Sal-Resistant Mutants Define Residues of RNAP Important for Function ofSal.

A total of 20 different substitutions conferring Sal-resistance wereidentified (Table 2). Substitutions were obtained at 11 sites in RNAP β′subunit (residues 690, 697, 738, 748, 758, 763, 779, 780, 782, and 783)and at 3 sites in RNAP β subunit (residues 569, 675, and 677) (Table 2;FIGS. 4A-C).

Seven additional Sal-resistant mutants were isolated followingmutagenesis of rpoC and rpoB. Substitutions conferring Sal-resistancewere obtained at 3 sites in RNAP β subunit (residues 561, 665, and 680)and 3 sites in RNA β′ subunit (residues 758, 780, and 782) (Table 3;FIGS. 4A-C).

In the three-dimensional structure of RNAP, the sites of substitutionsconferring Sal-resistance formed a tight cluster (the “Sal target”; seeFIG. 5). The identified Sal target was located immediately adjacent to,and partly overlapped, the RNAP active center. The Sal target waslocated adjacent to, but did not overlap, the targets of RNAP inhibitorsin current use in antibacterial therapy, rifamycins (see FIGS. 6; and10-15), and the target of an RNAP inhibitor under investigation for usein antibacterial therapy, CBR703 (see FIGS. 6; and 16,17).

The dimensions of the identified Sal target were ˜35 Å×˜18 Å×˜12 Å, andthus the identified Sal target was sufficiently large to be able toencompass Sal (˜16 Å×˜12 Å×˜10 Å). Based on the resistance propertiesand the size of the Sal target, it was inferred that the Sal target mostlikely was the binding site for Sal on RNAP.

TABLE 2 Spontaneous Sal-resistant mutants: sequences and propertiesnumber of amino acid independent resistance level substitution isolates(MIC/MIC_(wild-type))^(a) rpoC (RNAP β′ subunit) 690 Asn→Asp 2 16 697Met→Val^(b) 3 >16 738 Arg→Cys 2 >16 738 Arg→His 1 >16 738 Arg→Pro 2 >16738 Arg→Ser 1 >16 748 Ala→Glu 2 16 758 Pro→Ser 1 16 763 Phe→Cys 4 16 775Ser→Ala 1 8 779 Ala→Thr 2 >16 779 Ala→Val 5 >16 780 Arg→Cys 2 >16 782Gly→Ala 2 782 Gly→Cys 1 783 Leu→Arg 1 >16 rpoB (RNAP β subunit) 569Ile→Ser 2 >16 675 Asp→Ala 2 >16 677 Asn→His 1 >16 677 Asn→Lys 2 >16^(a)MIC with wild-type rpoC and wild-type rpoB was 0.024 μg/ml. ^(b)Oneisolate was a double-substitution mutant: 697 Met→Val; 1054 Thr→Ala.

TABLE 3 Induced Sal-resistant mutants: sequences and properties numberof amino acid independent resistance level substitution isolates(MIC/MIC_(wild-type))^(a) rpoC (RNAP β′ subunit) 758 Pro→Thr 1 4 780Arg→Cys 2 8 782 Gly→Cys 1 8 rpoB (RNAP β subunit) 561 Ile→Ser 1 2 665Ala→Glu 1 8 680 Leu→Met 1 4 ^(a)Assayed as D21f2toIC pRL663 andD21f2toIC pRL706 derivatives; MIC with wild-type rpoC and wild-type rpoBwas 0.098 μg/ml. ^(b)One isolate was a double-substitution mutant: 697Met→Val; 1054 Thr→Ala.

Crystal Structures Confirm Identification of Residues of RNAP Importantfor Function of Sal.

Crystal structures were determined for E. coli RNAP σ⁷⁰ holoenzyme inthe presence of Sal (resolution=4.2 Å) and in the absence of Sal(resolution=4.0 Å) (FIGS. 7A-B). Comparison of electron density maps forE. coli RNAP σ⁷⁰ holoenzyme in the presence of Sal to E. coli RNAP σ ⁷⁰holoenzyme in the absence of Sal revealed unambiguous difference densityattributable to Sal (FIG. 7A). The difference density was located in theSal target (FIG. 7A) and was in contact with or close to sites ofsubstitutions conferring Sal resistance are obtained (FIG. 7B). Theresults establish that the Sal target is the binding site on RNAP forSal, and that sites of substitutions conferring Sal-resistancecorrespond to RNAP residues of RNAP that contact or are close to Sal.

Resistance Levels of Sal-Resistant Mutants.

Resistance to Sal was quantified using broth microdilution assays. Allanalyzed mutants exhibited at least 2-fold resistance to Sal (Tables2,3). Thirteen mutants exhibited >16-fold resistance to Sal.

Cross-Resistance Levels of Sal-Resistant Mutants.

Cross-resistance to previously characterized small-molecule inhibitorsof RNAP was quantified by use of broth microdilution assays. TheSal-resistant mutants exhibited no cross-resistance with rifampin and nocross-resistance with CBR703 (Table 4). Indeed, two Sal-resistantmutants exhibited moderate, 2-fold, hypersensitivity to rifampin, andten Sal-resistant mutants exhibited moderate to high-level, 2-foldto >4-fold, hypersensitivity to CBR703 (mutants with resistance levels<1 in Table 4).

TABLE 4 Sal-resistant mutants: absence of cross-resistance to rifampinand CBR703 cross-resistance level amino acid (MIC/MIC_(wild-type))^(a)substitution rifampin CBR703 rpoC (RNAP β′ subunit) 690 Asn→Asp 1≦0.25^(b) 697 Met→Val 0.5 0.5 738 Arg→Cys 1 ≦0.25 738 Arg→Pro 1 ≦0.25738 Arg→Ser 1 1 748 Ala→Glu 1 ≦0.25 763 Phe→Cys 1 ≦0.25 779 Ala→Thr 1 1779 Ala→Val 1 ≦0.25 780 Arg→Cys 1 0.5 782 Gly→Ala 782 Gly→Cys rpoB (RNAPβ subunit) 569 Ile→Ser 1 1 675 Asp→Ala 0.5 ≦0.25 677 Asn→His 1 1 677Asn→Lys 1 ≦0.25 ^(a)MIC with wild-type rpoC and wild-type rpoB was 0.098μg/ml for rifampin and 6.25 μg/ml for CBR703. ^(b)Values <1 indicatethat the substitution conferred hypersensitivity to the inhibitor.

Cross-Resistance Levels of Rifamycin-Resistant Mutants.

More than 70% of clinical isolates of rifamycin-resistant Mycobacteriumtuberculosis contain β Asp516→Val, β His526→Asp, β His526→Tyr, or βSer531→Leu substitutions (15). Derivatives of E. coli D21f2tolCcontaining the corresponding rifamycin-resistant mutations were obtainedfrom laboratory stocks, and cross-resistance to Sal was assessed by useof broth microdilution assays. The rifamycin-resistant mutants exhibitedno cross-resistance to Sal (Table 5).

TABLE 5 Rifampin-resistant mutants: absence of cross-resistance to Salamino acid cross-resistance level substitution (MIC/MIC_(wild-type))^(a)rpoB (RNAP β subunit) 516 Asp→Val 1 526 His→Asp 1 526 His→Tyr 1 531Ser→Leu 1 ^(a)MIC with wild-type rpoB is 0.024 μg/ml.

Co-Administration of Sal and Rifampin Reduces Spontaneous Resistance toUndetectable Levels.

Spontaneous resistance frequencies were determined by plating E. coliD21f2tolC on LB agar (7) containing Sal at 4×MIC, rifampin at 4×MIC, orboth, and counting numbers of colonies after 24 h at 37° C. The resultsin Table 6 show that the spontaneous resistance frequencies for Salalone, rifampin alone, and Sal co-administered with rifampin were,respectively, 4×10⁻⁹, 1×10⁻⁸, and undetectable (<2×10⁻¹¹).

TABLE 6 Spontaneous resistance frequencies for Sal, rifampin, andco-administered Sal and rifampin spontaneous resistance compoundfrequency Sal 4 × 10⁻⁹ Rif 1 × 10⁻⁸ Sal + Rif <2 × 10⁻¹¹ 

Co-Administration of Sal and CBR703 Reduces Spontaneous Resistance toUndetectable Levels.

Spontaneous resistance frequencies were determined by plating E. coliD21f2tolC on LB agar (7) containing Sal at 4×MIC, CBR703 at 4×MIC, orboth, and counting numbers of colonies after 24 h at 37° C.

The results in Table 7 show that the spontaneous resistance frequenciesfor Sal alone, CBR703 alone, and Sal co-administered with CBR703 were,respectively, 4×10⁻⁹, 2×10⁻¹⁰, and undetectable (<2×10⁻¹¹).

TABLE 7 Spontaneous resistance frequencies for Sal, CBR703, andco-administered Sal and CBR703 spontaneous resistance compound frequencySal 4 × 10⁻⁹ CBR703  2 × 10⁻¹⁰ Sal + CBR703 <2 × 10⁻¹¹ 

Materials and Methods

Sal.

Sal was isolated as in reference 1.

Measurement of Nucleic Acid Synthesis In Vivo.

Macromolecular synthesis assays were performed essentially as inreference 5. Escherichia coli D21f2tolC (a strain with cell-envelopedefects resulting in increased susceptibility to antibiotics, includingSal; 6) was cultured in 10 ml M5T broth (5) at 37° C. with shaking untilOD₆₀₀=0.4-0.8, and cultures were diluted with pre-warmed M5T broth toOD₆₀₀=0.167. Aliquots (90 μl) were dispensed into wells of a 96-wellplate, were supplemented with 7 μl pre-warmed 6 ρCi/ml [¹⁴C]-uracil or[¹⁴C]-thymidine, were supplemented with 3 μl 0.048 μg/ml Sal in methanol(yielding a final Sal concentration two times the minimal inhibitoryconcentration) or 3 μl solvent blank, and were incubated at 37° C. withshaking. At time points 0, 5, 10, and 15 min after the addition of Salor solvent blank, rows of samples were transferred to a second 96-wellplate, containing 100 μl ice-cold 10% trichloroacetic acid (TCA) in eachwell, and the second plate was incubated on ice. One hour after thefinal time point, TCA precipitates were collected by filtration ontoglass-fibre filters (Filtermat A; Perkin-Elmer, Inc.; pre-rinsed twicewith 5% TCA), washed twice with 5% TCA, washed three times with water,and washed twice with 10% ethanol, using a Packard FilterMate 196 cellharvester with an OmniFilter upper head assembly (Perkin-Elmer, Inc.).Filters were dried under a heat lamp, wrapped in a single layer ofplastic wrap, and exposed to a phosphorimager screen for 16-18 h.Radioactivity was quantified using a Typhoon Variable Mode Imager andImageQuant v5 (Molecular Dynamics, Inc.).

Isolation of Sal-Resistant Mutants: Spontaneous Sal-Resistant Mutants.

E. coli D21f2tolC (6) was cultured to saturation in 5 ml LB broth (7) at37° C., cultures were centrifuged, and cell pellets (˜3×10⁹ cells) werere-suspended in 50 μl LB broth and plated on LB agar (7) containing 1.2μg/ml Sal (a concentration four times the minimal concentration requiredto prevent growth of wild-type cells under these conditions), andincubated 24-48 h at 37° C. Sal-resistant mutants were identified by theability to form colonies on this medium and were confirmed byre-streaking on the same medium.

Isolation of Sal-Resistant Mutants: Induced Sal-Resistant Mutants.

E. coli Random mutagenesis of rpoC in plasmid pRL663 and rpoB in plasmidpRL706 was performed as in 24, mutagenenized plasmids were passaged inE. coli XL1-Blue (Stratgene, Inc.) as in 24, mutagenized plasmids wereintroduced by transformation into E. coli D21f2tolC as in 24, andtransformants cells) were applied to LB-agar plates containing 1 μg/mlSal, 200 μg/ml ampicillin, and 1 mM IPTG, and plates were incubated16-24 h at 37° C. Sal-resistant mutants were identified by the abilityto form colonies on this medium and were confirmed by re-streaking onthe same medium.

PCR-Amplification and Sequencing of RNAP-Subunit Genes of Sal-ResistantMutants.

For spontaneous Sal-resistant mutants, rpoC and rpoB genese werePCR-amplified and sequenced as follows: Genomic DNA was isolated usingthe Wizard Genomic DNA Purification Kit (Promega, Inc.; procedures asspecified by the manufacturer) and was quantified by measurement ofUV-absorbance (procedures as in 7). The rpoC gene and the rpoB gene werePCR-amplified in reactions containing 0.2 ng genomic DNA, 0.4 μM forwardand reverse oligonucleotide primers

(5′-AGGTCACTGCTGTCGGGTTAAAACC-3′ (SEQ ID NO: 1)and 5′-TGACAAATGCTCTT TCCCTAAACTCC-3′(SEQ ID NO: 2) for rpoC; 5′-GTTGCACAAACTGTCCGCTCA ATGG-3′(SEQ ID NO: 3) and 5′-TCGGAGTTAGCACAATCCG CTGC-3′(SEQ ID NO: 4) for rpoB), 5 U TaqDNA polymerase (Genscript, Inc.), and 800 μM dNTP mix(Agilent/Stratagene, Inc.) (initial denaturation step of 5 min at 94°C.; 30 cycles of 30 s at 94° C., 45 s at 55° C., and 4.5 min at 72° C.;final extension step of 10 min at 72° C.). PCR products containing therpoC gene (4.3 kB) or the rpoB gene (4.1 kB) were isolated byelectrophoresis on 0.8% agarose (procedures as in 7), extracted from gelslices using the Gel/PCR DNA Fragments Extraction Kit (IBI Scientific,Inc.; procedures as specified by the manufacturer), and submitted toHigh-Throughput Sequencing Solutions (Seattle Wash.) for sequencing(Sanger sequencing; eight sequencing primers per gene).

For each induced Sal-resistant mutants, plasmid DNA was isolated andsubmitted to High-Throughput Sequencing Solutions (Seattle Wash.) forsequencing (Sanger sequencing; eight sequencing primers per gene).

Quantitation of Resistance to Sal.

Resistance to Sal was quantified by performing broth microdilutionassays. Single colonies were inoculated into 3 ml LB broth, andincubated 3-6 h at 37° C. with shaking. Diluted aliquots (2×10⁴ cells in98 μl LB broth; concentrations determined using OD₆₀₀=1 for 10⁹ cells)were dispensed into wells of a 96-well plate, were supplemented with 2μl of a 2-fold dilution series of Sal in methanol (final concentrationsof 1.56, 0.390, 0.195, 0.0975, 0.0488, 0.0244, 0.0122, and 0.00609μg/ml) or 2 μl of a solvent blank, and were incubated 16 h at 37° C.with shaking. The minimum inhibitory concentration (MIC) was defined asthe lowest tested concentration of Sal that inhibited bacterial growthby ≧95%.

Quantitation of Cross-Resistance to Rifampin and CBR703.

Cross-resistance levels were determined analogously to resistancelevels, using 0.12-0.78 μg/ml of rifampin (Sigma, Inc.) and 0.39-25μg/ml of CBR703 (Maybridge, Inc.).

Molecular Modeling.

Sites of substitutions conferring Sal-resistance were mapped onto acrystal structure of Thermus thermophilus RNAP holoenzyme (8; PDBaccession code 1L9U) and a crystal structure of the T thermophilustranscription elongation complex (RNAP in complex with DNA, RNA, and anucleoside triphosphate; 9; PDB accession code 205J). Correspondencesbetween residues of E. coli RNAP and T. thermophilus RNAP were based onamino acid sequence alignments (25; FIGS. 4A-C).

REFERENCES

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All publications cited herein are incorporated herein by reference.While in this application certain embodiments of invention have beendescribed, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art thatcertain of the details described herein may be varied without departingfrom the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar terms in thecontext of describing embodiments of invention are to be construed tocover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms “comprising,”“having,” “including,” and “containing” are to be construed asopen-ended terms (i.e., meaning “including, but not limited to”) unlessotherwise noted. Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. In addition to the orderdetailed herein, the methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate embodiments of invention and does not pose a limitation onthe scope of the invention unless otherwise specifically recited in theclaims. No language in the specification should be construed asindicating that any non-claimed element as essential to the practice ofthe invention.

What is claimed is:
 1. A method to treat a bacterial infection in asubject in need thereof, comprising administering to the subject a firstcompound selected as being an inhibitor of growth of a bacterium bybinding to the bridge-helix cap target of an RNA polymerase, and asecond compound that inhibits growth of a bacterium by binding to a siteother than the bridge-helix cap target of an RNA polymerase.
 2. Themethod of claim 1, wherein the first compound is salinamide A.
 3. Themethod of claim 1, wherein the second compound is a rifamycin or CBR703.4. The method of claim 3, wherein the second compound is rifampin orCBR703.
 5. The method of claim 1, further comprising administering athird compound that inhibits growth of a bacterium by binding to a siteother than the bridge-helix cap target of an RNA polymerase.
 6. Themethod of claim 1, wherein the first and second compounds areadministered concurrently.
 7. The method of claim 1, wherein the firstand second compounds are administered sequentially.
 8. The method ofclaim 5, wherein the third compound is administered concurrently withthe first or second compound, or concurrently with the first and secondcompound.
 9. The method of claim 5, wherein the third compound isadministered sequentially.
 10. A composition that comprises salinamide Aand a rifamycin and/or CBR703.
 11. The composition of claim 10 thatcomprises salinamide A and rifampin and/or CBR703.
 12. The compositionof claim 10 that comprises salinamide A and a rifamycin.
 13. Thecomposition of claim 12 that comprises salinamide A and rifampin. 14.The composition of claim 10 that comprises salinamide A and CBR703. 15.The composition of claim 10 that comprises salinamide A, a rifamycin,and CBR703.
 16. The composition of claim 15 that comprises salinamide A,rifampin, and CBR703.